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1 Supplementary Figures Supplementary Figure 1. nrg1 bns101/bns101 embryos develop a functional heart and survive to adulthood (a-b) Cartoon of Talen-induced nrg1 mutation with a 14-base-pair deletion in exon 2 (a); this deletion causes a frame shift leading to a premature stop codon in exon 2 resulting in a 1
2 predicted truncated protein (b). Notably, the predicted truncated Nrg1 protein in nrg1 bns101/bns101 animals lacks the Ig-like, EGF-like and TM domains that have all been reported to be essential for mouse cardiac trabeculation 1, 2, 3. If the first ATG downstream of the 14 bp deletion is used to reinitiate translation, a protein consisting of part of the TM domain and the carboxy terminal domain will be made. However, the type IIIa Nrg1 isoform 4 is not predicted to be affected by the bns101 mutation. (c-d) nrg1 bns101/bns101 larvae do not exhibit any gross morphological defects as compared to wild-type; lateral views, anterior to the left; scale bars, 0.5 mm. (e-h) Trabeculation appears unaffected in nrg1 bns101 mutants. 120 hpf larvae from Tg(myl7:LIFEACT-GFP); nrg1 bns101/+ incrosses imaged by spinning disk confocal microscopy; 2D views (mid-sagittal sections) (e and f), and 3D maximum intensity z-projections (g and h); V: ventricle; arrows point to trabecular cardiomyocytes; scale bars, 50 µm. 2
3 Supplementary Figure 2. Cardiac Nrg2a-mRFP expression during embryonic and larval development (a-i) Mid-sagittal confocal images of Tg(kdrl:NLS-EGFP);nrg2a +/- hearts; heat map; low intensity (blue) to high intensity(red) at 52 (a-c), 78 (d-f) and 120 (g-i) hpf; arrows and arrowheads point to endocardial cells in ventricular outer curvature and superior AV valve leaflet, respectively. (j) Graph showing that on average there is a higher percentage of endocardial cells positive for Nrg2a-mRFP in the ventricle than in the atrium at 78 hpf; dots in this graph represent individual hearts; N=5 hearts; values represent means ± SEM; ** P 0.01 by Student s t-test. (k) In situ hybridization for nrg2a expression in 78 hpf heart. V: ventricle, AV: atrioventricular canal, At: atrium; scale bars, 50 µm. 3
4 Supplementary Figure 3. Spatiotemporal pattern of cardiac Nrg2a-mRFP expression during embryonic and larval development (a-i) 3D views (maximum intensity z-projections) of Tg(kdrl:NLS-EGFP);nrg2a +/- hearts at 52 (a-c), 78 (d-f) and 120 (g-i) hpf; Nrg2a-mRFP expression appears stronger in the ventricle than in the atrium or AV canal; V: ventricle, AV: atrioventricular canal, At: atrium; scale bars, 50 µm. 4
5 Supplementary Figure 4. nrg2a -/- hearts develop functional atrioventricular valves (a-c) Maximum intensity z-projections (25 z-stacks, sagittal sections) of 120 hpf Tg(kdrl:NLS- EGFP);nrg2a +/- AV canal showing that Nrg2a-mRFP is weakly expressed in the AV valve leaflets. (d-i) Mid-sagittal confocal images of hearts from Tg(kdrl:NLS-EGFP);nrg2a +/- incrosses at 80 (d-f) and 120 (g-i) hpf; arrows and arrowheads point to superior and inferior valve leaflets, respectively; V: ventricle, AV: atrioventricular canal, At: atrium; scale bars, 50 µm. 5
6 Supplementary Figure 5. Developmental analysis of cardiac jelly thickness in the presence or absence of Nrg/Erbb2 signaling (a-h) Mid-sagittal views of wild-type and nrg2a -/- hearts from Tg(kdrl:Hsa.HRASmCherry);Tg(myl7:LIFEACT-GFP);nrg2a +/- incrosses at 55 (a-b), 60 (c-d), 72 (e-f) and 80 (gh) hpf; the myocardium and endocardium are labeled in green and red, respectively; magnified images of dashed boxes are shown below each time point. The thickness of the cardiac jelly progressively diminishes in wild-type and nrg2a -/- hearts. (i-k) Confocal sagittal sections of 82 hpf Tg(kdrl:Hsa.HRAS-mCherry);Tg(myl7:EGFP-Hsa.HRAS) hearts from wildtype (i), nrg1 -/- mutant (j) or Erbb2 inhibitor (PD168363) treated animals showing that cardiac jelly reduction also appears to be an Nrg1/Erbb2 independent process; magnified views of dashed boxes in i-h are shown on the right; arrows and asterisks indicate the presence and absence of cardiac jelly, respectively; V: ventricle, AV: atrioventricular canal, At: atrium; scale bars, 50 µm. 6
7 Supplementary Figure 6. Myocardial nrg2a overexpression causes cardiomegaly in wild-type fish (a-d) A number of injected F0 wild-type fish (6 out of 31) exhibited pericardial edema at juvenile and adult stages as compared to non-injected ones; lateral views of non-injected (a) and injected (c) zebrafish imaged by brightfield microscopy at 75 dpf; red arrowhead in c points to pericardial edema; scale bars, 1mm. 75 dpf extracted hearts from non-injected (b) and injected (d) fish; scale bars, 200 µm. 7
8 Supplementary Figure 7. Overexpression of nrg2a under control of a myocardial specific promoter induces cardiomyocyte bilayering in the ventricle by 46 hpf (a-b) 2D ventral views (mid-sagittal sections) of 46 hpf Tg(myl7:EGFP-Hsa.HRAS) (a) or Tg(myl7:EGFP-Hsa.HRAS);Tg(myl7:nrg2a-p2a-tdTomato) (b) hearts; Magnified images of dashed boxes are shown below a and b; arrows point to bilayered wall; V: ventricle, At: atrium; scale bars, 50 µm. 8
9 Supplementary Figure 8. Ectopic nrg2a overexpression in cardiomyocytes requires Erbb2 function (a-d) Confocal sagittal sections of Tg(myl7:EGFP-Hsa.HRAS) (a-b) or Tg(myl7:EGFP- Hsa.HRAS);Tg(myl7:nrg2a-p2a-tdTomato) (c-d) hearts, treated with DMSO (a and c) or the Erbb2 inhibitor PD (b and d) showing that nrg2a overexpression-induced cardiomyocyte multilayering requires Erbb2 function; asterisks and arrows indicate singlelayered and multilayered ventricular walls, respectively; V: ventricle, At: atrium; scale bars, 50 µm. 9
10 Supplementary Figure 9. Myocardial specific nrg2a overexpression leads to an increase in cardiomyocyte proliferation (a-d) 3D views (maximum intensity z-projections) of Tg(myl7:mVenus-gmnn) (a and c) or Tg(myl7:mVenus-gmnn);Tg(myl7:nrg2a-p2a-tdTomato) (b and d) hearts at 78 (a-b) and 96 (c-d) hpf. Proliferating cardiomyocytes are labeled in green; white and yellow dashed boxes outline the ventricular and atrial chambers, respectively; V: ventricle, At: atrium; scale bars, 50 µm. (e-f) Graphs showing the average number of proliferating cardiomyocytes per heart (e) and per ventricular or atrial (f) chamber at 78 and 96 hpf; N=9 hearts; values represent means ± SEM; * P 0.05,** P 0.01 by Student s t-test. 10
11 Supplementary Figure 10. Endocardial specific overexpression of nrg2a does not induce cardiomyocyte multilayering in tnnt2a morphants (a-b) 2D ventral views (mid-sagittal sections) of 96 hpf Tg(myl7:EGFP-Hsa.HRAS) (a) or Tg(fli1a:nrg2a-p2a-tdTomato);Tg(myl7:EGFP-Hsa.HRAS) (b) hearts from tnnt2a MO (0.5 ng) injected animals; magnified images of dashed boxes are shown below a and b; myocardium and endocardium are labeled in green and red, respectively; V: ventricle, At: atrium; scale bars, 50 µm. 11
12 Supplementary Figure 11. Nrg2a can affect cardiomyocyte behavior via both autocrine and paracrine signals (a-b) 2D ventral views (mid-sagittal sections) of 78 hpf Tg(myl7:EGFP-Hsa.HRAS) hearts from animals injected with tnnt2a MO (0.5 ng) and myl7:nrg2a-p2a-tdtomato DNA (5-10 pg). This low amount of DNA allows one to get single cardiomyocytes to overexpress the nrg2a transgene; arrows and arrowhead point to nrg2a-p2a-tdtomato expressing and nonexpressing cardiomyocytes, respectively; higher magnification of dashed box in a is shown in b; V: ventricle, At: atrium; scale bar, 50 µm. 12
13 Supplementary References 1. Erickson SL, et al. ErbB3 is required for normal cerebellar and cardiac development: a comparison with ErbB2-and heregulin-deficient mice. Development 124, (1997). 2. Kramer R, Bucay N, Kane DJ, Martin LE, Tarpley JE, Theill LE. Neuregulins with an Ig-like domain are essential for mouse myocardial and neuronal development. Proceedings of the National Academy of Sciences 93, (1996). 3. Lai D, et al. Neuregulin 1 sustains the gene regulatory network in both trabecular and nontrabecular myocardium. Circulation research 107, (2010). 4. Honjo Y, Kniss J, Eisen JS. Neuregulin-mediated ErbB3 signaling is required for formation of zebrafish dorsal root ganglion neurons. Development 135, (2008). 13
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